A comprehensive genetic catalog of human double-strand break repair.

The analysis of DNA sequence outcomes provides molecular insights into double-strand break (DSB) repair mechanisms. Using parallel in-pool profiling of Cas9-induced insertions and deletions (indels) within a genome-wide knockout library, we present a comprehensive catalog that assesses the influence of nearly every human gene on DSB repair outcomes. This REPAIRome resource uncovers uncharacterized mechanisms, pathways, and factors involved in DSB repair, including opposing roles for XLF and PAXX, a molecular explanation for Cas9-induced multinucleotide insertions, HLTF functions in Cas9-induced DSB repair, the involvement of the SAGA complex in microhomology-mediated end joining, and an indel mutational signature linked to VHL loss, renal carcinoma, and hypoxia. These results exemplify the potential of REPAIRome to drive future discoveries in DSB repair, CRISPR-Cas gene editing and the etiology of cancer mutational signatures. Editor's summary: When cells repair broken DNA, they can make mistakes that leave behind mutations, genetic scars that affect cell function and contribute to diseases such as cancer. DNA break–associated mutations also underlie CRISPR-based gene-editing outcomes. López de Alba et al. developed REPAIRome, a comprehensive catalog revealing how each of the approximately 20,000 human genes influences the mutation patterns arising from DNA double-strand break repair. REPAIRome offers insights into DNA repair mechanisms, enhances gene-editing precision, and explains the mutation patterns observed in cancer. In addition to being a tool for specialists in DNA repair, gene editing, or cancer evolution, this resource enables any researcher to explore whether a gene of interest affects DNA repair. —Di Jiang INTRODUCTION: DNA double-strand breaks (DSBs) are dangerous lesions whose incorrect repair can cause mutations and genome rearrangements linked to various diseases, including cancer onset and progression. DSBs also underlie the efficacy of a wide range of anticancer therapies, and their targeted induction and repair constitute the basis for gene editing technologies such as CRISPR-Cas systems. Understanding the molecular mechanisms of DSB repair therefore has direct implications for understanding tumor evolution and improving therapeutic and genome engineering strategies. RATIONALE: Sequence outcomes at DSB target sites in the form of insertions and deletions (indels) are not random but show specific patterns that define the relative contribution of the factors and pathways involved. Indel profiling in different contexts thus provides a potent tool for our molecular understanding of DSB repair mechanisms. In this study, we combined genome-wide CRISPR screening and massive-parallel profiling to provide a comprehensive picture of how Cas9-induced DSB-repair patterns are affected upon the disruption of each of more than 18,000 human genes. RESULTS: The REPAIRome resource that we generated provides a rich dataset in nontransformed (RPE1) and cancer (U2OS) cell lines that, with the help of a publicly available and browsable webtool (https://repairome.bioinfo.cnio.es/), can be consulted for the potential involvement of any gene of interest in DSB repair. Data-mining strategies based on differential comparison of repair profiles provided a comprehensive picture highly consistent with previous knowledge of nonhomologous end joining (NHEJ) and microhomology-mediated end joining (MMEJ) DSB repair pathways but also identified factors, mechanisms, and relationships, some of which were validated and further characterized during this study. These include (i) opposing roles for XRCC4-like factor (XLF) and paralog of XRCC4 and XLF (PAXX) paralogs in controlling DNA end processing to favor insertions and deletions, respectively; (ii) a mechanism of sequential multinucleotide insertions after Cas9 recutting of edited target sequences; (iii) the impact of helicase-like transcription factor (HLTF)–mediated removal of postcleavage Cas9 on repair outcomes; (iv) the involvement of the Spt-Ada-Gcn5 acetyltransferase (SAGA) chromatin-remodeling complex in MMEJ; and (v) the identification of insertional NHEJ as the molecular etiology of the ID11 cancer mutational signature, and its connection to von Hippel-Lindau (VHL) deficiency and hypoxia. CONCLUSION: With REPAIRome, we provide a consultative resource for the general research community, as well as a tool to drive scientific discoveries in the DSB repair, CRISPR-Cas gene editing, and cancer genomics fields, with its corresponding biotechnological and clinical implications. REPAIRome also sets a methodological framework for the high-throughput interrogation of molecular events associated with DSBs and potentially other targetable lesion types, with the capacity for being expanded and adapted to address additional questions regarding DNA repair mechanisms and their impact on genome instability. Overview of the REPAIRome results.: The scheme shows how our results are consistent with previous knowledge. DSB ends are filled in and repaired by NHEJ, resulting in insertions. Alternatively, after nucleolytic processing, DSBs can be repaired by MMEJ or NHEJ. A network representation highlights the factors identified in the REPAIRome screen as being involved in each of these steps within the DSB repair process. Specific examples of novel insights obtained in this study are indicated [(i) to (v)], as described in the Structured Abstract. [ABSTRACT FROM AUTHOR]